A protein incorporating a non-natural amino acid (hereinafter also referred to as an “alloprotein”) in which an amino acid residue at a desired position in a protein is replaced with an amino acid other than 20 different amino acids involved in normal protein synthesis (a non-natural amino acid) could offer an effective means of analyzing the function and structure of a protein. Meanwhile, lysine derivatives include amino acids, such as acetyl-lysine, methyl-lysine etc., which are synthesized by post-translational modification. Such amino acids are well-known particularly as those involved in regulation of gene expression by histones and are also known as those involved in regulation of transcriptional activation, regulation of protein-protein interaction, and suppression/promotion of ubiquitination for many types of proteins. It is expected that many findings concerning acetylation, methylation etc. of lysine could be made if those lysine derivatives could be introduced site-specifically into a protein synthesized by a eukaryote.
Pyrrolysyl tRNA synthetase (PylRS) is a novel aminoacyl tRNA synthetase (aaRS) found in a methanogenic archaebacterium (Methanosarcina). A corresponding tRNA (pyrrolysine tRNA) is a suppressor tRNA, which has a unique secondary structure such as an unusually small D loop, etc. Recently, it was found that in Escherichia coli, PylRS and pyrrolysine tRNA do not interact with endogenous aaRS and tRNA (orthogonality), and pyrrolysine could be introduced specifically into the site of an amber codon in a protein (Non-Patent Document 1). Further, it has been reported that a wild-type PylRS can bind a non-natural amino acid such as Nε-Boc-L-lysine to pyrrolysine tRNA in Escherichia coli (Non-Patent Document 1).
On the other hand, in a mammalian cell, enzymes that phosphorylate tyrosine residues in proteins (tyrosine kinases) play an important role in transducing extracellular signals, such as by growth stimulating factors, into the nucleus. The tyrosine kinases include one capable of phosphorylating a tyrosine derivative and one incapable of phosphorylating a tyrosine derivative. For example, it was shown that a Src kinase phosphorylates an iodotyrosine residue but an EGF receptor cannot do so. Thus, it is useful in examining interaction of a desired protein with various tyrosine kinases in a cell if an alloprotein, the desired protein into which a tyrosine derivative is incorporated, could be synthesized in a mammalian cell. For example, it is important in analysis of signal transduction mechanisms to examine which tyrosine kinase phosphorylates the desired protein. Further, these non-natural amino acid-incorporated proteins could be useful in themselves as material for analysis of the function and structure of a protein, and could have a novel bioactivity.
As an expression method of an alloprotein like the above in an animal cell, there has been developed a method of expressing in an animal cell (A) a mutant tyrosyl tRNA synthetase (hereinafter referred to as “mutant TyrRS”), which is a variant of a tyrosyl tRNA synthetase derived from Escherichia coli and has an increased specificity to a non-natural tyrosine derivative as compared with the specificity to a tyrosine, (B) a suppressor tRNA originating from eubacterium, such as bacillus, mycoplasma, and staphylococcus, and capable of binding to the above tyrosine derivative in the presencGe of the above mutant tyrosyl tRNA synthetase, and (C) a desired protein gene including a nonsense mutation or frame shift mutation at a desired position. The above tyrosine derivative has been incorporated into the position of the nonsense mutation or frame shift mutation of the above protein (Patent Document 1 and Non-Patent Document 2).
Hereupon, it is required that the above suppressor tRNA originating from the non-eukaryote is transcribed by an RNA polymerase in a eukaryotic cell. In contrast to one kind of RNA polymerase in prokaryotic cells, it is known that in eukaryotic cells, there are three different kind of RNA polymerases I, II, and III (polI, polII, and polIII) that share functions. PolI synthesizes ribosomal RNA, PolII synthesizes mRNA, and PolIII synthesizes 5S rRNA, tRNA, U6 small nuclear RNA (snRNA), etc. Therefore, tRNA in a eukaryotic cell is synthesized by transcription by RNA polymerase III. Genes transcribed by the RNA polymerase III are classified broadly into three groups according to characteristics of their promoter structures, the groups including, as their representative genes, a 5S rRNA gene (Type I promoter), a tRNA gene (Type II promoter), and a U6 small nuclear RNA gene (Type III promoter), respectively. The type II promoter, which transcribes a tRNA, is an internal promoter made up of two regions in a tRNA coding sequence, the consensus sequences of which are known as box A and box B. The consensus sequence of the box A consists of the positions 8-19: TRGCNNAGYNGG (SED ID NO:1), and the consensus sequence of the box B consists of the positions 52-62: GGTTCGANTCC (SED ID NO:2). Accordingly, for example, the suppressor tyrosine tRNA of Bacillus stearothermophilus, although it originates from a prokaryote, can be expressed in an animal cell without any alterations, because of the presence of the box A and box B in its suppressor tyrosine tRNA coding sequence (refer to Non-patent Document 3, for example).
Here, incorporation of an amino acid into the position of the nonsense mutation in the above protein is referred to as suppression. Because there are only three different types of stop codons, a maximum of three types of non-natural amino acids can be incorporated into one type of protein. In vitro experiments have developed artificial base pairs in addition to naturally occurring base pairs (refer to Non-patent Documents 4 and 5), and an RNA containing artificial base pairs as mentioned above can be transcribed in vitro by using an RNA polymerase of T7 bacteriophage. It is expected that the following could be achieved: increase in the number of codon types, which are now 43, by using artificial base pairs in codons encoding amino acids, and introduction of a plurality of non-natural amino acids into one type of protein by getting the codons that do not encode natural amino acids to encode non-natural amino acids.    [Patent Document 1] WO2004/039989A1    [Non-Patent Document 1] Blight, S. K. et al., Nature, 431, 333-335 (2004)    [Non-Patent Document 2] Sakamoto, K. et al., Nucleic Acids Research 30, 4692-4699 (2002)    [Non-Patent Document 3] M. Sprinzl et al., Nucleic Acids Research 17, 1-172 (1989)    [Non-Patent Document 4] Hirao, I. et al., Nature Biotechnology 20, 177-182 (2002)    [Non-Patent Document 5] Hirao, I. et al., Nature Methods 3, 729-735 (2006)